Help with Multiplex Nested PCR - (Aug/14/2010 )

I have a Multiplex Nested PCR in the lab where it was standardize few years ago by a student in his master degree.
In this technique we can amplify a highly conservated region (NS5) from Flavivirus using Universal Primers for genus and then with Multiplex Nested PCR, using internal primers species-specific we can distinguish each other.
The problem is: sometimes it works fine, but sometimes it doesn't.
We make two step: RT step using MMLV (invitrogen) and followed by PCR with Taqman (invitrogen) and run electrophoresis and see if there's some positive sample or not. If so, we perform the Multiplex Nested PCR.
In RT step i use reverse primer under 65ºC for 5 min, followed by chilling on ice for 5 minutes then add MMLV, buffer and DTT and cycle in 37°C for 60 min, 70ºC 15 min to inactivated MMLV
In PCR step i use Forward primer , making a mix with all adding 5 ul of cDNA template from RT and cycle: 94ºC for 3 minutes, 94ºC for 1 min, 53ºC for 1 min and 72ºC for 2 minutes for 35 cycles and after all 72ºC for 5 minutes.

The fragment amplified may be around 984kb. The problem is sometimes we can see this amplicon, but sometimes not
Is it possible, this amplicon would be visualized only in Multiplex nested PCR?
In your opnion is there something wrong in PCR protocol?
What about MMLV? Is it a good RT enzime?

Thank you!

-Richard.21-

Hi!
My first question if you are trying to improve your method without significantly changing any step. I would think that your problem is robusteness and yield.
Dou you quantify your RNA before retrotranscribing it?
Why do you choose to do specific priming in RT, it improves your specificity? Have you compared random priming vs specific priming?
Your first product is pretty large, and I would think this step is rate limiting if you start with variable amount of cDNA. Try to increase by 3-4 the number of cycles and see if it is enough Taq polymerase and free magnesium in the reaction (If everything is ok, 1U and 2 mM would do. But sometimes you need to increase enzyme (and Mg2+/dNTP) accordingly, depending on the complexity of your substrate). This can give you a more robust first step, but you will have to be careful with specificity in the nested step!.
MMLV should do, if it is working in your lab. I particularly like engineered enzymes due to their increased thermal stability. However, they are more expensive, this is a cost-benefit relationship.
Hope my insight can be useful
Rocio

-Rociohache-

Hi!

Yes i would like to improve this method, but sometimes it doesn't work in the first steps. I changed all reagents, got new primers, and even today it didn't work. Positive control is a viral stock (clearly have a lot of RNA available) and even this has not amplified.
I don't quantify my RNA before RT......
I don't know if random primers would be more efficient in this RT step, because my samples are from clinical samples from cerebrospinal fluid, where the viral load is very low.... what do you thing about Random primer?
Someone told me that in clinical samples Random primers works good to optime and increase PCR sensibility. What do u think?
What about increase RT step of 37ºC for 2 or 3 hours?
And what about Superscript III? Is it good too?

Thank youy very much again

Rociohache on Mon Aug 16 17:33:05 2010 said:

Hi!
My first question if you are trying to improve your method without significantly changing any step. I would think that your problem is robusteness and yield.
Dou you quantify your RNA before retrotranscribing it?
Why do you choose to do specific priming in RT, it improves your specificity? Have you compared random priming vs specific priming?
Your first product is pretty large, and I would think this step is rate limiting if you start with variable amount of cDNA. Try to increase by 3-4 the number of cycles and see if it is enough Taq polymerase and free magnesium in the reaction (If everything is ok, 1U and 2 mM would do. But sometimes you need to increase enzyme (and Mg2+/dNTP) accordingly, depending on the complexity of your substrate). This can give you a more robust first step, but you will have to be careful with specificity in the nested step!.
MMLV should do, if it is working in your lab. I particularly like engineered enzymes due to their increased thermal stability. However, they are more expensive, this is a cost-benefit relationship.
Hope my insight can be useful
Rocio